Abstract
We have examined the imprecision in the estimation of PCR efficiency by means of standard curves based on strategic experimental design with large number of technical replicates. In particular, how robust this estimation is in terms of a commonly varying factors: the instrument used, the number of technical replicates performed and the effect of the volume transferred throughout the dilution series. We used six different qPCR instruments, we performed 1–16 qPCR replicates per concentration and we tested 2–10μl volume of analyte transferred, respectively. We find that the estimated PCR efficiency varies significantly across different instruments. Using a Monte Carlo approach, we find the uncertainty in the PCR efficiency estimation may be as large as 42.5% (95% CI) if standard curve with only one qPCR replicate is used in 16 different plates. Based on our investigation we propose recommendations for the precise estimation of PCR efficiency: (1) one robust standard curve with at least 3–4 qPCR replicates at each concentration shall be generated, (2) the efficiency is instrument dependent, but reproducibly stable on one platform, and (3) using a larger volume when constructing serial dilution series reduces sampling error and enables calibration across a wider dynamic range.
Highlights
Literature search (e.g. Pubmed) for scientific publications using the keyword “quantitative PCR” retrieves hundreds of thousands of hits, manifesting that qPCR has become mainstream life sciences technology [1,2,3]
The aim of this study is to test the impact of three experimental factors on the precision of the estimated PCR efficiency: (1) the effect if qPCR instrument changes; (2) the effect of how many technical replicates are included; and (3) the effect of the volume transferred across dilutions
We tested three experimental factors in terms of precision of the estimated PCR efficiency: (1) the effect of the qPCR instrument; (2) the impact of the number of technical replicates; and (3) the effect of the volume transferred across dilutions
Summary
Literature search (e.g. Pubmed) for scientific publications using the keyword “quantitative PCR” (qPCR) retrieves hundreds of thousands of hits, manifesting that qPCR has become mainstream life sciences technology [1,2,3]. It is widely acknowledged as the most sensitive method to quantify minute amounts of nucleic acids and its applications split into two main types referred to as: relative [4,5] and absolute [6,7,8] quantification. In spite of these heroic efforts, the standard curve remains the most reliable and robust approach to estimate PCR assay efficiency that is broadly accepted by the community [16], while some of the alternative approaches have found use as quality control tools in high-throughput setups [17,18,19]
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